DESCRIPTION: In this revised application, Dr. Beutler proposes a comprehensive study of various red cell enzyme deficiencies, including polymorphisms of the TPI gene (AIM3 of original proposal). Two specific aims are now described: In AIM1 PI proposes to deduce structure/function relationships underlying deficiencies of enzyme function in the most common genetic abnormalities of erythrocyte metabolism: Mutations affecting glucose-6-phosphate dehydrogenase (G6PD), pyruvate kinase (PK), glucose phosphate isomerase (GPI), and triosephosphate isomerase (TPI). The specific gene mutation underlying each case of red cell enzyme deficiency will be identified by restriction digest (in the case of known recurring mutations) and by direct sequencing of genomic DNA, where recurring mutations are unlikely. The mutant proteins will then be produced in E. coli or in K-562 cells using recombinant tag-expression systems, in order to circumvent various problems associated with purification from red cell. Wild type and mutant enzymes will then be compared with respect to Km for substrate and enzyme stability. Where possible, reference to the crystal structure of the enzyme (or, related structure) will be made. The crystal structure of PK will be pursued through collaboration with Dr. Varughese. In AIM2, Dr. Beutler proposes to identify the TPI mutation that is common in the African-American population and believed to be lethal when homozygous. Preliminary data suggest deficiency is related to point mutation at positions -5,-8 and/or -24 from the transcriptional start site, although the impact of these identified polymorphisms on gene expression has not been confirmed. PI will continue to accumulated blood samples from Dr. Schneider (Chicago Medical School) for TPI enzyme assay, and for analysis of potential mutations in the 5' untranslated region of the gene that might affect transcription. Initial effort will focus on the known polymorphisms at -5, -8, and -24 nt from the transcriptional start site. The impact of these mutation on gene expression will be tested in vitro using reporter constructs. Should these point mutations fail to alter gene transcription, a search will be made for other potential mutations in linkage disequilibrium with the known -5,-8 and -24 polymorphisms, by sequencing upstream through the promotor.